This invention relates to optical detection instruments more particularly optical detection systems that may illuminate, collect and detect light of a broad spectral range of wavelengths.
In chemical, biochemical and cellular analytical systems, imaging provides a powerful tool. The imaging may use fluorescence, luminescence (e.g. chemiluminescence) or radio labels in a large number of analytical procedures. This imaging allows very sensitive detection. The use of such labels is well characterized, for example as detection agents conjugated to probes or binding agents. Alternatively the label may be directly incorporated into a target of interest. The sensitivity of these labels allows detection of rare events and rapid sample analysis. Optical analysts may be applied to a number of different analytical applications including identification of compounds, assay of array of biomolecules (e.g. biopolymers) or assay of binding events (e.g. competitive binding).
The benefits of imaging technology have led to widespread application of this technology to research needs. One benefit of optical screening has been in increased throughput. The speed and sensitivity of optical scanning is adaptable to automated high throughput assays that are required for screening large numbers of samples against numerous interaction agents.
Developments in the fields of genomics, cytology and chemistry have produced challenges for the evaluation and analysis of large numbers of unique samples, compounds or biological isolates.
For example, in the field of genetics, the human genome project and other genome sequencing efforts have identified tens of thousands of unique oligonucleotide sequences. Screening of gene expression against these known sequences would provide information on gene expression and regulation. This information could be correlated with disease states or applied to evaluate response of cells to therapeutic treatment (e.g. for clinical evaluation of potential pharmaceutical evaluation, monitoring of response to therapy, etc.). Labeled mRNA or cDNA isolated from cells could be used in expression evaluations. Alternatively, genomic fragments may be screened against known sequences to determine homology between the screened DNA and known genes.
Clinical evaluation of DNA expression could require analysis of DNA from hundreds of thousands of individuals. This analysis could involve screening of nucleic acid isolations from hundreds of different cell types against tens of thousands of unique oligonucleotide sequences. The present need for throughput has motivated the development of multiplexed, automated, high throughput analysis of nucleic acid homology.
There is no single, standardized technology for nucleic acid analysis. In part this is a result of the differing throughput needs. The density of analytical arrays or the scale of separation analysis of DNA will depend on the amount of samples that are being analyzed. Gene expression analysis may be performed in wells on a multiwell plate, on a separation gel, on a chip substrate containing an array of reaction spots or using other formats. If a separation gel or chip substrate is used, the separation substrate may either be read directly or transferred to another substrate, (e.g. a transfer [blotting] membrane). Alternatively, an emitted signal from an assay of samples (e.g. chemiluminescent signal, radio signal) may be recorded on a storage device (e.g. a storage phosphor screen), which is subsequently analyzed. The samples of interest may be detected by detecting a signal associated with the sample.
In addition to the label and substrate variability, the sample size may vary. This may range from the relatively large target from a separation gel to a spot on a chip array, which may be orders of magnitude smaller. Presently, most automated analytical systems are limited in types of substrates and target densities that the system is able to analyze. The data from different analytical systems often must be subsequently combined to make a finalized evaluation of a biological sample or compound.
High throughput analysis ability is required to analyze sample nucleic acids using known sequences to study genetic variation, differential expression, etc. High throughput systems have taken advantage of automation to increase sample analytical rate. Optical analytical systems may further increase sample processing throughput by designing systems with multiplex capability. For example, the ability to distinguish multiple dyes at a single dye location allows a number of dyes, each associated with a unique probe or binding agent to be used in a single assay. The combination of markers may be used at a single target loci and distinguished by the analytical system by the unique dye emission wavelength profile. This further increases the information gained from each analytical scan and increases analytical throughput. However, this would only be possible in an analytical system in which the illumination and detection optics were adapted for use with a number of different emission profiles and could operate over a wide spectral range.
In a number of scanning fluorescent analytical instruments, the excitation light and the generated fluorescent emission share a pathway and must be optically separated. Some mechanism is needed to separate an excitation laser beam from a spectrally shifted collinear, counter propagating collected fluorescent light. Typical systems use a dichroic mirror to reflect wavelengths of the laser light and transmit wavelengths of the emission light. One example is seen in Baer et al. (U.S. Pat. No. 5,547,849). A laser produces coherent light that is directed onto a dichroic mirror. The mirror directs light of the wavelengths produced by the laser through an objective lens and onto a sample. The focused beam waist is directed onto a layer to be scanned. The emitted fluorescent light is collected by the objective lens and transmitted as a collimated retrobeam to the dichroic mirror. The mirror is selected to transmit the wavelengths of the collected fluorescent light to detection optics. An alternative system is seen in Kamentsky (U.S. Pat. No. 5,072,382). In this system a laser produces an illumination beam that is directed through a dichroic mirror and onto a focus lens. The focus lens focuses the illumination light onto a sample, exciting fluorescence. The lens collects excited fluorescent light, which is transmitted as a retrobeam to the dichroic mirror. Light of the fluorescent wavelengths is reflected by the mirror onto detection optics.
These arrangements allow the rapid scanning of a sample and detection of emitted light. However, these systems have several significant drawbacks. First, the coatings on dichroic mirrors may be angle sensitive when the coatings must filter or reflect multiple sets of wavelengths. A slight variation in angle can result a significant change in the wavelengths transmitted and reflected by the mirror coating. In such cases, the positioning of the mirror must be precise for proper functioning. Second, the coatings are designed to transmit and reflect specific spectral bands. This severely limits the illumination and emission wavelengths that may be used in the optical scanning system. Changing the excitation laser wavelength or the fluorescent dye (having an alternative emission profile) would require replacement of the dichroic mirror. For any one dichroic mirror, the system is locked into specific wavelength choices. Third, the coatings that are designed to transmit or reflect multiple excitation wavelengths must still be selected for specific wavelength bands. It is common that the excitation wavelength will encroach on an emission profile of a fluorescence dye. Since the dichroic mirror transmits only a selected range of wavelengths, some emission intensity outside of this range is lost. The separation of excitation and emission wavelengths may result in a substantial loss of the collected fluorescence due to the dichroic mirror not transmitting some emission light to the detectors.
Versatility in analyzing various analytical substrates is also important to maximize the utility of optical analytical systems. Presently, a number of different substrates are used to analyze bio-molecular samples. These include gels, microplates, membranes, arrays (disposed on plastic, glass or membranes) storage phosphor screens storing radiant energy images from various samples and other devices. At present, optical analytical systems are generally designed for analysis of a single type of device. The elements of an analytical device, such as the reading stage and the focusing optics are generally selected to be used with a single sample type. In many optical analytical systems fixed illumination optics determine an illumination geometry, limiting the range of targets sizes which may be illuminated by a single system. As applied this has meant that different analytical systems must be used for different analytical targets. An analytical system would have increased utility if the system were adaptable to a range of target sizes and target densities. This would allow a single analytical system to scan a number of different sample types and sample densities.
A number of different optical readers have been developed to provide technology to meet imaging needs. U.S. Pat. No. 6,043,506 describes an optical scanner in which optical fibers are fixed into a scan head for illumination and/or collection of light from a sample. The scan head is moved by a transfer gantry to scan the head in two dimensions over a sample surface. To ensure the scan head is operating within the desired parameters of the system, the scan head is moved to a separate calibration location prior to each scan. Although some control of the resolution of the scanning system is possible (e.g. by limiting the scanning geometry by finer graduated steps of the scan head), the fixed optics of the scan head provides a limit to the resolution of the scan system.
U.S. patents granted to Mathies et al. are also relevant to the field of the present invention. In U.S. Pat. No. 4,979,824, an optical analytical apparatus is described. This apparatus is based on a flow cytometry system and utilizes a spatial filter to define a small probe volume that allows for detection of individual fluorescent particles and molecules. Laser power and exposure time of the sample are selected to enhance signal-to-noise ratio. Real-time detection of photon bursts from fluorescent particles is used to distinguish the number, location or concentration of the particles from background energy.
In U.S. Pat. No. 5,091,652 to Mathies et al., a laser-excited fluorescent scanner is enclosed for scanning separated samples using a confocal microscope. Generally, the sample is separated on a slab gel by electrophoresis. The gel is subsequently optically analyzed. Alternatively, the sample may be analyzed on a membrane, filter paper, petri dish, or glass substrate. The confocal microscope directs an illumination laser beam into the sample with beam oriented so that background scattering is minimized by the polarization characteristics of the scattered light.
U.S. Pat. No. 5,274,240 also granted to Mathies et al. and a continuation-in-part of the above patent, teaches a capillary array scanner. This invention is primarily intended for fluorescence detection from a plurality of in-line capillary tubes containing samples that have been separated by capillary electrophoresis. The fluorescence detection assembly employs a confocal system to detect fluorescence from the interior volumes of each capillary tube.
U.S. Pat. No. 5,784,152 discloses an optical scanner for analyzing multiwell plates, gel plates, or u storage phosphor screens. The sample is illuminated with focused light that is xe2x80x9ctunedxe2x80x9d by filters to a selected illumination wavelength. The detection is also xe2x80x9ctunedxe2x80x9d by filters or other optical devices to allow for light detection at a selected wavelength. For both illumination and detection a bandpass filter is used as a primary wavelength selector. As with the prior referenced system, the system resolution is both fixed and fairly limited. The plate or gel is scanned by two-dimensional movement of the stage or illuminating light.
U.S. Pat. No. 5,591,981 discloses a method and apparatus that provides continuous tuning of excitation and/or emission in fluorescent imaging. For both illumination and detection, dispersive elements or filters are used to produce spectral shifts to transmit a selected wavelength range. A non-coherent, broad wavelength lamp is focused to illuminate the sample. The emitted light passes through a filter on a filter wheel and an interferometer. The light emission then passes onto a charge coupled diode detector. The detection system is tuned to different settings to allow individual, one-at-a-time detection of individual dyes.
In these optical systems, fixed sample scanning optics limit the system to relatively narrow ranges of detection resolution.
The scanning mechanism of the optical system impacts the throughput potential of the system. Movement of a scan head requires additional scan time, requires additional calibration and requires additional motor parts that increase system size. Transmission and collection of multiple wavelengths in optical fibers limits resolution. The optical transmission fiber diameter must be sufficient for multimode propagation if multiple wavelengths are transmitted through the fiber. Light may lose coherence when the light is propagated through multiple fibers. If the wavelengths are not aligned into various fibers (due to varying fiber length) the beam may lose Gaussian properties. In some systems, the use of long pass filters in conjunction with each detector limits each detector to detect a single wavelength intensity measurement.
It is an object of the invention to provide a reader for the optical analysis of a number of different substrates, including chip arrays, microplate wells, cells within microplate wells, gels and storage phosphor screens. It is a further object to allow the illumination and detection to be selectable across a number of wavelengths. It is a further object of the invention to be able to detect at a plurality of resolutions, including detection of discrete targets 1-100 um in width. It is a further object to enable detection of a greater number of dyes than detection channels used.
The objects are achieved with an optical analytical system that scans in a limited depth of field. The system allows illumination and detection at selectable wavelengths while also allowing for selectable resolution of detection. In this system, a selected set of one or more lasers provide excitation illumination. If multiple illumination sources are used, the beams may be optically combined into a single illumination beam or used individually. Separate optics split the illumination beam, diverting part of the beam into a beam detector that monitors beam output and power fluctuation. A selectable laser line filter is used with each laser to filter out wavelengths other than the selected laser wavelength.
The illumination beam may be directed through a zoom contractor/expander optic that shapes the beam spot. This multi-element lens is focused to shape the illumination beam, producing a beam spot of a selectable size at the targeted sample location. The beam spot size is selected to concentrate energy into a limited depth. This would allow optimized scanning of different density arrays. After passing through the beam expander/contractor optic, the beam is directed by a scanning optic that produces a beam scan in a first direction dimension. A galvo mirror, resonant scanner, rotating polygonal mirror, or acousto/optic scanner, or other scanning optic may be used to effect the beam scan. The focused scanned beam is directed onto the sample.
The emitted light is collected and sent to the detection optics. The objective acts as a wide angle light collector that collects light and transmits the light as a retrobeam to the detection optics. The excitation beam and emission light are separated using an optical element having a central optic, that directs the illumination light to the sample, surrounded by an annular optic that directs the collected emission light to the detection optics. This could be effected by a central broadly reflective mirror annularly surrounded by a transmissive material that allows collected emission light to pass to the detection optics. In another embodiment, a central hole in a broadly reflective mirror allows the illumination beam to pass through the illuminate the sample. The collected fluorescent light beam is radially much larger than the illumination beam. The collected retrobeam is reflected by the mirror onto detection optics.
In the present invention the beam focusing optics allow the system to be used to analyze a range of sample densities present on a number of different types of substrates. In one embodiment, the arrays are present on the bottom of multiwell plates. Autofocus mechanisms allow rapid focus onto the bottom of multiwell plates or other analytical substrates. Alternatively, storage phosphor screens may be scanned. The storage phosphor screen records the signal emitted from either a luminescent or radioactive label.